The present invention relates to retroviral vectors. The invention particularly relates to retroviral vectors that have an enhanced 3xe2x80x2 transcription termination structure and to methods for using such vectors to express heterologous coding sequences in mammalian cells and organisms.
Retroviral Vectors
Retroviral vectors are currently one the most frequently used gene delivery vehicles in gene therapy protocols. Fundamental to the utility of retroviral vectors is the various retrovirus characteristics retained by the vectors. Such characteristics include efficient transfection of many cell types and stable integration of their genomes into a host cell chromosome, which enables long-term expression of vector encoded genes. Another important retained characteristic is that the initial steps of the vector life cycle from binding of vector particles through integration of its genome into a host cell""s genetic material require no de novo synthesis of viral proteins.
Basic Components of Retroviral Vectors
The main features of the wild-type retroviral genome are summarized in FIG. 1, which shows the open reading frames and the structures of the viral long terminal repeats (LTRs). Retroviral vectors comprise genomes derived from retroviruses. The simplest type of retroviral vectors have a significantly pared down retroviral genome which is missing most of the sequences encoding viral genes (e.g., gag, env and pol) and retains only sequences that are required for the packaging, reverse transcription and integration. The pared down retroviral genomes are often referred to as retroviral backbones, upon which further modifications can be made and to which heterologous genes and sequences can be added to form retroviral vectors. Typically, a heterologous gene is inserted into the backbone in such a way that allows the 5xe2x80x2 LTR promoter to drive its subsequent expression. An expression construct comprising a heterologous gene operatively associated with a promoter can also be inserted into the backbone for delivery and expression in a target cell.
Retroviral vectors missing some or all of the viral genes are replication deficient. Production of viral particles comprising such vectors requires vector propagation in host cells that provide the missing functions in trans. Trans complementation can be achieved in various ways including transfecting the host cell with a packaging helper construct, also derived from a retroviral genome, which expresses the missing viral proteins but cannot be packaged because of a deletion of the packaging signal. This system of retroviral vector production is illustrated in FIG. 2. When both the vector and packaging helper construct are present in a producer cell, infectious retroviral particles are released that are capable of delivering the vector genome with its inserted gene. This process of gene delivery is referred to as transduction.
Lentiviral Vectors
To date, the most common retroviral vectors used in clinical gene therapy protocols have been based on the murine leukemia virus (MuLV), and a variety of packaging systems to enclose the vector genome within viral particles have been developed (reviewed in Miller, A D. 1997. Development and applications of retroviral vectors. In Retroviruses, Ed. Coffin J M, Hughes S H, Varmus H E. CSHL Press, New York.). The vectors themselves have all of the viral genes removed, are completely replication-defective, and can accept up to approximately 6-8 kb of exogenous DNA. These current vector/packaging systems seem to pose minimal risk to patients, and to date there have been no reports of toxicity or long-term problems associated with their use.
However, MuLV and vectors derived from it are only able to infect dividing cells. This is because the pre-integration nucleoprotein complex is unable to cross an intact nuclear membrane. In contrast, the prototypical lentivirus HIV-1 has been shown capable of nuclear import even when an intact membrane exists, and HIV-1-derived vectors are therefore able to transduce non-dividing cells (Naldini et al., Science 272:263-267 (1996)). This property of HIV vectors makes them particularly attractive candidates for gene therapy when the target cell is non-dividing and stable integration of the heterologous gene is required.
Improvements in Retroviral Vector Design and Production Systems
The retroviral vector production system described above is functional but unsatisfactory in several ways. In particular, overlaps that remain between the vector sequences and sequences encoding viral components in packaging helper constructs means that there is a significant risk of recombination events that would create an infectious replication-competent retrovirus (RCR). Such overlaps exist largely because extensive sequences of the gag gene are retained in the vector to enhance packaging efficiency. In addition, the LTRs are frequently retained in packaging helper constructs to provide both promoter and polyadenylation sequences.
In order to minimize the risk of RCR production, various improved approaches to vector design and production have been developed. One approach splits the packaging components, placing the gag-pol genes and the env gene onto separate plasmids that can be individually introduced into the packaging cell. In another approach, Env-mediated recombination is avoided by the use of heterologous envelope proteins whose coding sequences have no homology with the genome of the parental retrovirus but which can be incorporated into the vector particle (a process referred to as pseudotyping). A commonly used heterologous envelope protein is VSV G, the G protein from vesicular stomatitis virus (Burns et al., Proc. Natl. Acad. Sci. 90:8033-8037 (1993)). See FIG. 3, Panel A.
In yet another approach, LTR-mediated recombination is reduced by the use of heterologous promoters and polyadenylation signals in the packaging helper constructs. This can also have the advantage of enhancing vector titer (Soneoka et al., Nucleic Acids Res 25:628-633 (1995)). This approach typically involves deleting non-essential sequences from the vector LTRs and where appropriate, replacing the deleted sequences with heterologous sequences. For example, heterologous promoters, such as the CMV immediate-early promoter, have been used to replace the 5xe2x80x2 U3 promoter. In other instances, 3xe2x80x2 U3 sequences have been significantly deleted, as is the case with self-inactivating (SIN) vectors, as long as the integrase recognition sequences (i.e., att sequences) are retained (Yu et al., Proc. Natl. Acad. Sci 83:3194-3198 (1986)). See FIG. 3., Panel B.
These approaches have been used in developing various lentivirus-based vectors, which raise special safety concerns because of the possibility of pathogenic RCR arising from recombination events. Example products of this approach include the CMV-driven SIN vectors (Zufferey et al., J. Virol. 72:9873-9880 (1988)), the minimal packaging helper constructs with all of the non-essential genes inactivated or removed (Zufferey et al., Nat. Biotechnol. 15:871-875 (1997)), and retroviral particles comprising non-HIV-1 envelope proteins such as the VSV G.
Retroviral Vector Integration
Retroviral vectors integrate their genomes into a host cell""s genetic material. A great deal is known about the process of retroviral integration, which is carried out by the viral integrase. Integrase recognizes sequences at the ends of the LTRs of the DNA provirus (the att sites, FIG. Panel 1B), and inserts the provirus more or less randomly into the host genome, although some sequence preferences have been reported (Carteau et al., J. Virol. 72:4005-4014 (1988)).
The ability of retroviral vectors to integrate is a two-edged sword. On the one hand, it allows for the possibility of stable long-term expression of vector encoded genes, with the integrated provirus being passed on to all daughter cells. On the other hand, vector integration can interfere with the normal functioning of flanking host genes. Indeed, retroviruses were first identified on the basis of their ability to cause oncogenic transformation. One type of interference is the inappropriate activation of host genes by read-through transcription, i.e. the continuation of viral transcripts past the 3xe2x80x2 LTR transcription termination site and into downstream host gene sequences. Read-through transcription from proviruses into host sequences has been observed for several retroviruses (R. V. Gunataka Microbiol. Rev. 57:511-521 (1993); Bohnlein et al., J. Virol. 63:421-424 (1989); Herman et al., Science. 236:845-848 (1987); Iwasaki et al., Genes and Dev. 4.2299-2307 (1990); Cherrington et al. EMBO J. 11:1513-1524 (1992)). Recent evidence shows that integrated HIV-1 provirus can also effect read-through transcription of flanking host cell sequences (Dron et al., Arch Virol. 144:19-28 (1999)).
Transcription Termination of mRNAs
The 3xe2x80x2 end of messenger RNAs (mRNAs) transcribed by RNA polymerase II is created by cleavage of the nascent transcript. This event occurs predominantly at a polyadenylation site and is followed by the template-independent addition of an approximately 250-nucleotide poly(A) tail (Wahle et al., FEMS Microbiol. Rev. 23:277-295 (1999)). It has been suggested that the poly(A) tail influences many aspects of mRNA metabolism, including stability, translational efficiency, and transport of processed mRNA from the nucleus to the cytoplasm (Lewis et al., Microbiol Mol Biol Rev. 63:405-445 (1999); Colgan et al., Genes and Dev. 11:2755-2766 (1997); Huang et al., Mol. Cel. Biol. 16:1534-1542 (1996); Sachs et al., J. Biol. Chem. 268: 22955-22958 (1993)). A strong polyadenylation signal has been observed to increase the level of precursor cleavage and the length of poly (A) of mRNA produced in vitro (Lutz et al., Genes and Dev. 10:325-337 (1996)). In one instance, increased poly (A) tail length correlates with enhanced transgene expression (Loeb et al., 1999 West Cost Retrovirus Meeting, abstract p57).
Polyadenylation Signals
The core polyadenylation signal consists of two recognition elements flanking a cleavage/polyadenylation site. A highly conserved AAUAAA hexanucleotide element (Proudfoot et al., Nature 263:211-214 (1976)) is located 8 to 31 nucleotides upstream of the cleavage site (Chen et al., Nucleic Acid Res. 23:2614-2620 (1995)) and a poorly conserved GUxe2x80x94 or U-rich (G/U-rich) downstream element is located 14 to 70 nucleotides downstream of the AAUAAA element. Cleavage of the mRNA transcript usually occurs after an A residue, with a preference for a CA dinucleotide, between these two elements (Sheets et al., Nucleic Acid Res. 18:5799-5805 (1990))(FIG. 4).
A growing number of polyadenylation signals have also been shown to contain additional elements located upstream of the AAUAAA sequence that enhance transcription termination (FIG. 4). Early examples of such upstream enhancers (UEs) were found in the polyadenylation signals of various viruses, including HIV-1 (Valsamakis et al., Mol. Cell. Biol. 12:3699-3705 (1992); Gilmartin et al., EMBO J. 11:4419-4428 (1992)), equine infectious anemia virus (Graveley et al., J. Virol. 70:1612-1617 (1996)), simian virus 40 (SV40) (Carswell et al., Mol. Cell. Biol. 9:4248-4258 (1989)), adenovirus (Prescott et al., Mol. Cell. Biol. 14:4682-4693 (1994); DeZazzo et al., Mol. Cell. Biol. 9:4951-4961 (1989)), cauliflower mosaic virus (Sanfacon et al., Genes and Dev. 5:141-149 (1991)), and ground squirrel hepatitis virus (Cherrington et al., J, Virol. 66:7589-7596 (1992)) poly(A). More recently, UEs have also been identified in the polyadenylation signals of mammalian genes, such as the human complement C2 gene (Moreira et al., EMBO J. 14:3809-3819 (1995); Moreira et al., Genes and Dev. 12:2522-2534 (1998)) and the lamin B2 gene (Brackenridge et al., Nucleic Acid Res. 25:2326-2335 (1997)).
In general, UEs comprise U- or UG-rich sequences, but there is no clear sequence homology between different UEs (Carswell et al., Mol. Cell. Biol. 9:4248-4258 (1989); R. H. Russnak, Nucleic Acid Res. 19:6449-6456 (1991); Sanfacon et al., Genes and Dev. 5:141-149 (1991); Moreira et al., EMBO J. 14:3809-3819 (1995)) (FIG. 5). Notwithstanding the absence of sequence homology, certain viral UEs appear functionally interchangeable (Russnak et al., Genes and Dev. 4:764-776 (1990); Valsamakis et al., Proc Natl Acad Sci USA. 88:2108-2112 (1991); Graveley et al., J. Virol. 70:1612-1617 (1996)).
The UE of the SV40 late polyadenylation signal (also known as USE) is located 13-51 nucleotides upstream of the MUAM element (FIG. 5) (Schek et al., Mol Cell Biol. 12:5386-5393 (1992); Lutz et al., Genes and Dev. 8:576-586 (1994); Carswell et al., Mol. Cell. Biol. 9:4248-4258 (1989); Cooke et al., Mol Cell Biol. 19:4971-4979 (1999)). The USE plays an important role in enhancing the activity of the core polyadenylation signal as USE mutations reduced polyadenylation efficiency by 75 to 85%, both in vitro and in vivo (Carswell et al., Mol. Cell. Biol. 9:4248-4258 (1989); Schek et al., Mol. Cell. Biol. 12:5386-5393 (1992)). Within the USE, three core U-rich elements with the consensus sequence AUUUGUPuA have been identified as the active components. They apparently function in a distance-dependent manner, and when present in multiple copies, in an additive manner on polyadenylation efficiency (Carswell et al., Mol. Cell. Biol. 9:4248-4258 (1989)). The UE of the ground squirrel hepatitis virus polyadenylation signal also influences the activity of the core polyadenylation signal in a orientation-dependent, additive but distance-independent manner (R. H. Russnak, Nucleic Acid Res. 19:6449-6456 (1991)).
Retroviral Polyadenylation Signals
Retroviral 5xe2x80x2 and 3xe2x80x2 LTRs contain a polyadenylation signal AAUAAA in the R region, a G/U-rich downstream element is located in the U5 region and the cleavage/polyadenylation site defines the R/U5 boundary (FIG. 4). In HIV-1, the 3xe2x80x2 LTR has an UE (also known as UHE) in the U3 region, 77-94 nucleotides upstream of the AAUAAA motif (FIG. 4, Panel C, and FIG. 5). The UHE significantly increases the processing efficiency of the 3xe2x80x2 LTR polyadenylation signal (DeZazzo et al. Mol Cell Biol. 12:5555-5562 (1991); Valsamakis et al., Mol Cell Biol. 12:3699-3705 (1992)). A putative minor polyadenylation enhancer, designated UHEM, has also been identified 146-171 nucleotides upstream of the AAUAAA motif (Valsamakis et al., Proc Natl Acad Sci USA. 88:2108-2112 (1991)) (FIGS. 4 and 5).
Despite the presence of the AAUAAA and G/U-rich downstream elements at both the 5xe2x80x2 and 3xe2x80x2 LTRs, the HIV-1 polyadenylation signal copied from the 3xe2x80x2 LTR is preferentially recognized. Several mechanisms have been proposed to account for the differential recognition of the two polyadenylation signals (Das et al., J. Virol. 73:81-91 (1999); Cherrington et al., J, Virol. 66:7589-7596 (1992); DeZazzo et al., Mol Cell Biol. 12:5555-5562 (1992); J. Cherrington, EMBO J. 11:1513-1524 (1992)).
The present invention relates to retroviral vectors that have an enhanced 3xe2x80x2 transcription termination structure. In one embodiment, retroviral vectors of the invention comprise one or more heterologous upstream enhancer (UE) sequences operably associated with the 3xe2x80x2 LTR polyadenylation signal. In another embodiment, retroviral vectors of the invention comprise additional copies of endogenous UE sequences operably associated with the 3xe2x80x2 LTR polyadenylation signal. The invention provides compositions comprising such retroviral vectors, their nucleotide sequences, viral particles produced by such vectors, and cells comprising such vectors and their proviral sequences. The invention also provides methods for using such retroviral vectors for expressing heterologous coding sequences in mammalian cells and organisms.
The present invention is based on the surprising discovery that incorporating one or more heterologous UE sequences, or one or more additional copies of endogenous UE sequences into retroviral vectors increased the transcriptional termination efficiency of their 3xe2x80x2 LTR, and that vectors having such modification produced higher vector titers (i.e., titers of viral particles comprising the vector) than those produced by otherwise identical vectors having no such modifications. While not intending to be limited to any theory, it is believed that enhancing transcriptional termination by the 3xe2x80x2 LTR increases the production, stability, nuclear export and/or translation of vector mRNA, and that such increases lead to higher vector RNA production and/or gene expression, and hence the higher vector titers in producer cells.
Definitions
Unless otherwise specified herein, the following words and terms shall have the following meanings with respect to the present disclosure and the appended claims.
xe2x80x9c3xe2x80x2 LTRxe2x80x9d refers to a 3xe2x80x2 retroviral long terminal repeat, which may or may not be modified from its corresponding native (i.e., that existing in the wild-type retrovirus) 3xe2x80x2 LTR by deleting and/or mutating endogenous sequences and/or adding heterologous sequences.
xe2x80x9c5xe2x80x2 LTRxe2x80x9d refers to a 5xe2x80x2 retroviral long terminal repeat, which may or may not be modified from its corresponding native 5xe2x80x2 LTR by deleting and/or mutating endogenous sequences and/or adding heterologous sequences.
xe2x80x9c3xe2x80x2 LTR polyadenylation signalxe2x80x9d refers to the polyadenylation signal present in the 3xe2x80x2 LTR of retroviruses.
xe2x80x9cUpstream enhancerxe2x80x9d and xe2x80x9cUExe2x80x9d are used interchangeably, and refer to a control element present in the 3xe2x80x2 untranslated region of various eukaryotic and viral genes that enhances transcriptional termination by a polyadenylation signal located downstream of the enhancer. Examples of UEs are found in the SV40 late polyadenylation signal (USE), the HIV-1 LTR (UHE) and the ground squirrel hepatitits virus (UGE).
xe2x80x9cUpstream enhancer sequencexe2x80x9d and xe2x80x9cUE sequencexe2x80x9d are used interchangeably, and refer to the sequence of a UE or an active segment thereof. Like a UE, an active segment of a UE increases the transcriptional termination activity of a polyadenylation signal when it is placed 5xe2x80x2 upstream of that signal. A UE may comprise many active segments that may or may not be overlapping in sequence.
In the context of the retroviral vectors of the invention, a xe2x80x9cheterologousxe2x80x9d UE sequence is a UE sequence from a UE not identical to the one present in the native 3xe2x80x2 LTR of the retrovirus from which the retroviral vector of the invention is derived. By contrast, an xe2x80x9cendogenousxe2x80x9d UE sequence is a UE sequence from a UE present, such as UHE of HIV-1, in the native 3xe2x80x2 LTR of the retrovirus from which the retroviral vector of the invention is derived.
xe2x80x9c3xe2x80x2 transcription termination structuresxe2x80x9d of a retroviral vector refer to structures within and proximal to the 3xe2x80x2 LTR that effect termination of transcriptions initiated upstream of the structures. Such structures comprise the 3xe2x80x2 LTR polyadenylation signal and may additionally comprise endogenous UE sequences and heterologous UE sequences operatively associated with that signal.
xe2x80x9cPolynucleotidexe2x80x9d refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
xe2x80x9cProducer cellxe2x80x9d refers to a cell that comprises a retroviral vector or its proviral sequence and produces transducing particles comprising the retroviral vector. Where the retroviral vector is replication deficient, the producer cell complements the deficiency by producing the required replication function(s) in trans.
xe2x80x9cRetrovirusxe2x80x9d denotes a class of viruses that use RNA-directed DNA polymerase, or xe2x80x9creverse transcriptasexe2x80x9d to copy a viral RNA genome into a double-stranded DNA intermediate which integrates into the chromosomal DNA of a host cell. Retroviruses include lentiviruses. Examples of retroviruses include, but are not limited to, Moloney murine leukemia virus, spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumour virus. Examples of lentiviruses include human immunodeficiency virus, simian immunodeficiency virus, equine infectious anemia virus, feline immunodeficiency virus, visna virus.
xe2x80x9cVectorxe2x80x9d refers to xe2x80x9cretroviral vector,xe2x80x9d unless otherwise specified.
xe2x80x9cRetroviral vector genomexe2x80x9d refers to a polynucleotide comprising sequences from a retroviral genome that are sufficient to allow an RNA version of that polynucleotide to be packaged into a retroviral particle, and for that packaged RNA polynucleotide to be reverse transcribed and integrated into a host cell chromosome by the action of the retroviral enzymes, such as reverse transcriptase and integrase, contained in the retroviral particle.
xe2x80x9cGenexe2x80x9d refers to a polynucleotide that encodes a polypeptide.
xe2x80x9cCoding sequencexe2x80x9d refers to a polynucleotide that encodes a polypeptide, antisense RNA, a ribozyme or a structural RNA, such as snRNA, tRNA and rRNA.
In the context of the retroviral vectors of the invention, a xe2x80x9cheterologousxe2x80x9d gene or coding sequence is a gene or coding sequence that is not identical to any gene or coding sequence found in the retrovirus from which the retroviral vector of the invention is derived.
Two genes or sequences are xe2x80x9cidenticalxe2x80x9d if the order of nucleotides in each gene or sequence is the same, without any addition, deletion or material substitution.
In the context of polynucleotides, a xe2x80x9csequencexe2x80x9d is an order of nucleotides in a polynucleotide in a 5xe2x80x2 to 3xe2x80x2 direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polynucleotide.
xe2x80x9cOperatively associatedxe2x80x9d refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For example, a UE sequence is operatively linked to a polyadenylation signal in the same DNA molecule if the UE sequence enhances transcriptional termination by that signal. Similarly, a promoter is operatively associated with a coding region in the same DNA molecule if the promoter enables transcription of the coding sequence. There may be intervening residues between such associated elements so long as their functional relationship is maintained.